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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 10 - May 17, 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed in compliance with the Good Laboratory Practice (GLP) regulations (revised in 1997, ENV/MC/CHEM(98)17). The method followed that described in the OECD Guidelines for Testing of Chemicals, Updated Guideline No 406 Skin Sensitisation (adopted 17 Julyl 1992).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The data have been generated before teh OECD 429 protocol was mandatory for REACH.

Test material

Constituent 1
Chemical structure
Reference substance name:
1,​1'-​Biphenyl, 4-​(trans-​4-​ethylcyclohexyl)​-​2,​3',​4',​5'-​tetrafluoro-
EC Number:
605-735-6
Cas Number:
174805-87-9
Molecular formula:
C20 H20 F4
IUPAC Name:
1,​1'-​Biphenyl, 4-​(trans-​4-​ethylcyclohexyl)​-​2,​3',​4',​5'-​tetrafluoro-
Test material form:
solid
Specific details on test material used for the study:
Batch: E96635863
Purity: 99,9% (HPLC)
Feb 28, 1998

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: HsdPoc:DH
Sex:
female
Details on test animals and environmental conditions:
Test System: Guinea pig, HsdPoc:DH, females
Breeder: Charles River Kisslegg
Age: about 7 weeks

The mean initial body weight at the start of the study was 400 g (range from 362 to 446 g).

In a large number of tests, with a variety of test materials, the guinea pig and the specific strain used has proven to be a species well suited for skin sensitization studies.

Identification and adaption
Healthy young animals were allocated to the study groups at least 7 days before dosing to allow them to acclimatize. The guinea pigs were identified by colour mark, whereas the pretest animals were marked with a different colour than the animals of group 1 and 2 (details are documented in the raw data).
Assignment
30 guinea pigs were used in this study.
Group 1: negative control group (5 females + 5 males)
Group 2: test material group (10 females + 10 males)

Housing:
The guinea pigs were housed in a 10 m2 room of the Test Institute. Lighting was controlled by a timer to provide a 12-hour light - 12-hour dark regime.
Five guinea pigs were housed in type GM/5 (EBECO) Makrolon cages with a shelter and placed on mobile racks. The animals were kept on conventional softwood granulate as the
bedding. The cages had been machine cleaned before the start of the study. The bedding was changed two times a week.
Temperature and atmospheric humidity were measured by a thermohygrograph. The room temperature within the study period was 23 to 26°C and the relative atmospheric humidity 45 to 65 %.
Diet and community tap water from Makrolon drinking bottles were available to the guinea pigs ad libitum. The diet, Altromin Standard Diet, was checked according to the specifications of the
manufacturer by independent laboratories. Analysis included both qualitative and quantitative evaluation for heavy metals, aflatoxins, pesticides and antibiotics. The tap
water was analyzed periodically according to the German regulations for human drinking water. The softwood granulate was analytically checked by independent laboratories.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
other: intradermal and topical
Vehicle:
paraffin oil
Concentration / amount:
see details on study design
Challengeopen allclose all
Route:
other: topical
Vehicle:
paraffin oil
Concentration / amount:
see details on study design
No. of animals per dose:
Control group: 10 (5/5)
Test group 20 (10/10)
Details on study design:
PRE TEST
To determine the concentrations suitable for the main study, a pretest with single intradermal or topical administrations of the vehicle and of test material preparations was performed. Intradermal (i.d.) injections were given to one animal. Topical (top.) applications were given to another two animals.
In the pretest the following concentrations were used:
1. Liquid paraffin ( vehicle) intradermal: undiluted
2. Test material: intradermal.: 50, 25, 10, 5, and 1 g/L paraffin
topical.: 250, 100, 50, and 10 g/L paraffin


MAIN STUDY
A1. INDUCTION EXPOSURE (intradermal injection)
After shaving of the shoulder region, six intradermal injections were given (three on each
side of the spinal column in a total area of 2 x 4 cm). The guinea pigs received the
following injections of 0.1 ml each:

Group 1 (vehicle: Liquid paraffin)
cranial: 0.05 mL Freund's complete adjuvant + 0.05 mL sodium chloride solution
medial: 0.1 mL Liquid paraffin
caudal: 0.05 mL Freund's complete adjuvant + 0.05 mL sodium chloride solution

Group 2 (test material)
cranial: 0.05 mL Freund's complete adjuvant + 0.05 mL sodium chloride solution
medial: 0.1 mL (25 g test material/L ad liquid paraffin)
caudal: 0.1 mL Freund's complete adjuvant + sodium chloride solution (25 g test material/L preparation)


A2. INDUCTION EXPOSURE (topical application)
One week after the intra.dermal injections, the shoulder area of the guinea pigs was shaven
again and on injection sites covered with a filter paper patch of about 8 cm2, fully soaked with 1 ml of the test material preparation or the vehicle. The patches were attached for 48
hours under occlusive conditions.

Group 1: Liquid paraffin
Group 2: Test material (100 g/L ad Liquid paraffin)


B. CHALLENGE EXPOSURE
Two weeks after the topical induction the challenge was performed by fixing filter papers of about 4 cm2 fully loaded with 0.5 ml of the test material preparations (10 g/l liquid paraffin) to the left and soaked with 0.5 ml liquid paraffin (undiluted) to the right shaven flank ofthe animals. The patches were fixed for 24 hours with occlusive plastic tape.


Observation schedule

Clinical investigations
During the induction phase the guinea-pigs were examined daily for local skin reactions, and the results were documented. The challenge sites were investigated for reactions 48 hours after start of the
challenge. Further inspections followed 72 hours after start of the challenge, to detect weak or slowly developing reactions.

Following the grading according to the EEC Directive 2001/59/EEC, a result in an adjuvant method is considered positive if 30% or more ofthe test animals showed positive
reactions.

Body Weight
The body weight of the guinea-pigs was determined prior to the start of the study, then on day 8, 15, 22, and at the end ofthe study.
Positive control substance(s):
yes
Remarks:
DNCB

Results and discussion

Positive control results:
50% positive reactions with control substance

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25 g/L (intradermal induction I), 100 g/L (topical induction II), 10 g/L (topical challenge)
No. with + reactions:
5
Total no. in group:
20
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
25 g/L (intradermal induction I), 100 g/L (topical induction II), 10 g/L (topical challenge)
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 g/L induction, 0 g/L challenge
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
0 g/L induction, 0 g/L challenge
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
other: challenge control
Dose level:
Induction included intradermal injection of the test material formulation (25 g/L with and without FCA) on day 1, and topical application of the test material formulation (100 g/L) on day 8, challenge with liquid paraffin
No. with + reactions:
4
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
other: challenge control
Dose level:
Induction included intradermal injection of the test material formulation (25 g/L with and without FCA) on day 1, and topical application of the test material formulation (100 g/L) on day 8, challenge with liquid paraffin
No. with + reactions:
3
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.1 g/L DCNB
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
positive control
Dose level:
0.1 g/L DCNB
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the given experimental conditions, the test material induced no reactions. According to EU Regulation No. 1272/2008 (CLP), the test material is not classified as a skin sensitizer.
Executive summary:

Purpose

The purpose of this GPMT assay was to identify the contact allergenic potential of teh test material. This study should provide a rational basis for risk assessment to the sensitising potential of the test item in man.

Conclusion

Under the given experimental conditions, the test material induced no reactions. According to EU Regulation No. 1272/2008 (CLP), the test material is not classified as a skin sensitizer.

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